Combination immunotherapy targeting LAG-3, PD-1 and STING suppresses hepatocellular carcinoma as monitored by LAG-3 targeted PET imaging

Abstract Background The low response rate of anti-PD-1 monoclonal antibodies (mAbs) in hepatocellular carcinoma (HCC) requires the development of combination immunotherapy strategies to improve their efficacy. This study aimed to use LAG-3-targeted PET imaging to monitor the efficacy of anti-PD-1 mAb, a stimulator of interferon genes (STING) agonist, and anti-LAG-3 mAb, both individually and in combination. Furthermore, we evaluated the potential of a triple immunotherapy regimen (anti-PD-1 mAb, STING agonist, and anti-LAG-3 mAb) to improve HCC treatment. Methods The LAG-3 inhibitor C25 based on a cyclic peptide was chelated with NOTA, radiolabeled with 68 GaGaCl 3 . The resulting 68 GaGa-NOTA-C25 underwent in vivo PET imaging and ex vivo biodistribution examination in Hepa1-6 tumor-bearing mice. 68 GaGa-NOTA-C25 PET was used to monitor the efficacy of monotherapy and dual immunotherapy with anti-PD-1 monoclonal antibody (mAb) and STING agonists. The tumor uptake of 68 GaGa-NOTA-C25, tumor response, and survival rates were measured following different treatments. The therapeutic efficacy, molecular mechanisms, and safety of triple immunotherapy were validated using histopathological analysis and flow cytometry. Results 68 GaGa-NOTA-C25 PET imaging effectively and noninvasively detected LAG-3 + tumor-infiltrating lymphocytes (TILs) in Hepa1-6 tumor-bearing mice. In mice treated with anti-PD-1 mAb, STING agonist, or a combination immunotherapy, 68 GaGa-NOTA-C25 PET revealed significantly increased LAG-3 + TIL levels. At the treatment endpoint, the combination of the STING agonist with the anti-PD-1 mAb resulted in a significantly higher uptake (1.35 ± 0.191%ID/g) compared to the control group (0.402 ± 0.017%ID/g), the anti-PD-1 mAb group (0.647 ± 0.037%ID/g), and the STING agonist group (0.874 ± 0.089%ID/g). Uptake of 68 GaGa-NOTA-C25 was positively correlated with tumor therapeutic effects and survival rates. Triple immunotherapy with anti-PD-1 mAb, a STING agonist, and anti-LAG-3 mAb further enhanced efficacy compared to any dual immunotherapy regimen, and treatment efficacy was linearly associated with 68 GaGa-NOTA-C25 tumor uptake. Conclusions Anti-PD-1 mAb and STING agonists have shown notable synergy in upregulating LAG-3 expression on TILs in HCC, which can be successfully tracked by 68 GaGa-NOTA-C25 PET imaging. Furthermore, integration of a triple immunotherapy regimen comprising an anti-PD-1 mAb, STING agonist, and anti-LAG-3 mAb demonstrated a significant improvement in therapeutic efficacy over dual immunotherapy approaches.